As a species, we are distinguished from other primates by our capacity for language, our ability to walk upright, and our talent for inventing and using sophisticated tools. These traits originated in physical adaptations, such as increased brain size and changes in the morphology of the limbs, which required changes in development. Although it has long been thought that changes in gene regulation drove the evolution of uniquely human traits, in vivo evidence for human-specific developmental regulatory functions remains elusive. Moreover, despite intensive efforts to annotate functional elements in the genome, such as the ENCODE project, there is no parallel effort to annotate uniquely human cis-regulatory functions. The goal of this study is to identify and characterize developmental enhancers with human-specific functions in vivo and examine their role in human evolution.
The aims of our proposal are based on extensive preliminary data. We have developed a novel computational approach to identify noncoding, potentially regulatory sequences that are highly conserved across vertebrate species, but that changed substantially during human evolution. Using a mouse transgenic enhancer assay, we have identified eight of these "human-accelerated" conserved noncoding sequences (HACNSs) that function as enhancers during development. Of particular relevance for the aims of this proposal, we have also shown that the most rapidly evolving element in our dataset, HACNS1, is a developmental enhancer that has gained a robust limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This domain includes the presumptive anterior wrist and proximal thumb. Here we will build on these previous studies to generate more sophisticated maps of human-specific sequence acceleration in noncoding elements, including transcription factor binding site data and ChIP-seq data generated by genome-wide efforts to annotate regulatory function. We will use these data in conjunction with computational and experimental filters, such as extreme evolutionary constraint and recruitment of p300, to predict enhancers and mouse transgenic technologies to identify and characterize enhancers with human-specific activities. We will use synthetic enhancers and biochemical approaches to determine how human-specific sequence change alters enhancer function. Finally, we will use a reverse genetic strategy to study the evolutionary relevance of the human-specific functional change in HACNS1, by using gene targeting to replace the mouse ortholog of HACNS1 with the human enhancer and evaluating the effect of this genetic change on mouse development. The results from these studies will complement and extend current efforts to functionally annotate the human genome and will begin to reveal the precise molecular evolutionary events that produced modern humans.

Public Health Relevance

Understanding the genetic basis of human morphology and development is central to the public health mission of the NIH. Beyond the fundamental question of human origins, studying human evolutionary history is directly relevant to human health, as many common human diseases may in part be due to the effects of sequence changes that were advantageous early in our evolution but are now harmful. Evidence for this hypothesis stems from human and chimpanzee comparisons: many prevalent human diseases with a genetic component, including skin cancers, heart disease, malaria infection, Alzheimer disease, multiple sclerosis and major psychoses, appear to be uncommon, different or absent in chimpanzees. Our research may thus shed light on the genetic and evolutionary basis of diseases that are unique to our species.